1. Field of the Invention
This invention relates to X-ray crystallography, and, in particular, to methods and apparatus for mounting and aligning of samples for X-ray crystallographic analysis.
2. Discussion of the Art
X-ray crystallography is an established, well-studied technique for providing a three-dimensional representation of the appearance of a molecule in a crystal. Scientists have employed X-ray crystallography to determine the crystal structures of many molecules.
In order to perform an X-ray crystallographic analysis, a sample of the crystal must be mounted onto a positioning device, then carefully aligned so that the entire crystal is within the diameter of the X-ray beam, and X-ray diffraction data collected at a number of rotational angles. Because the typical sizes of crystals and the diameter of the X-ray beam are in the range of 100 to 400 micrometers, the alignment requires a high degree of precision. In addition, to ensure the integrity of crystals, the crystals must be stored under liquid nitrogen and maintained at temperatures near that of liquid nitrogenduring the entire mounting, aligning, and data collecting processes. Currently, mounting and aligning of samples is performed manually.
A typical X-ray crystallography apparatus comprises an X-ray generator, a detector, and a rotating spindle onto which a finely adjustable head of the positioning device is mounted. Raw diffraction data collected by the detector are input into a computer for processing. The head of the positioning device allows minute adjustments in two axes that are perpendicular to one another and to the axis of the spindle. Some heads of positioning devices also allow for angular input into a computer for processing. The head of the positioning device allows minute adjustments in two axes that are perpendicular to one another and to the axis of the spindle. Some heads of positioning devices also allow for angular adjustments in one or more axes. A third axis of adjustment is provided by translation of the rotating spindle in a direction that is orthogonal to the two axes of the head of the positioning device. The sample mount position of the head of the positioning device is positioned so that when mounted, the sample is near the centerline of the X-ray beam. A CCD camera is mounted so that a magnified image of the mounted sample can be displayed on a video monitor. Cross-hairs on the video display indicate the desired position of the sample, corresponding to the intersection of the center of the X-ray beam with the axis of the spindle. In order to maintain the sample at a sufficiently low temperature once it is mounted, a stream of cold nitrogen gas is directed at the sample mount position.
The procedure for mounting and aligning a sample manually is described below. An operator places a sample into a small canister of liquid nitrogen and then maneuvers the canister near to the sample mount position on the head of the positioning device. As quickly as possible, the operator withdraws the sample and mounts it onto the head of the positioning device. Using the video image on the monitor, the operator turns adjustment screws controlling the xe2x80x9cXxe2x80x9d, xe2x80x9cYxe2x80x9d, and xe2x80x9cZxe2x80x9d axes until the sample is centered within the X-ray beam and spindle axes (as indicated by the cross-hairs on the video display). After the sample has been centered, analysis of the sample by X-ray diffraction is begun. The procedure is described in detail in Garman, et al., xe2x80x9cMacromolecular Cryocrystallographyxe2x80x9d, J. Appl. Cryst. (1997) 30, 211-237 (hereinafter xe2x80x9cGarman et al.xe2x80x9d), incorporated herein by reference.
According to Garman et al., there are numerous problems involved in manual procedures for X-ray-diffraction data collection from macromolecular crystals at cryogenic temperatures. According to Garman, prerequisites for starting a cryogenic data collection are a reliable cryostat, the ability to maintain an ice-free environment, some crystal-mounting equipment, a sufficient number of crystals, and some manual dexterity for smooth and rapid operation on the part of the operator. An important part of a cryocrystallographic data collection is the method of crystal mounting and the hardware associated with it. Macromolecular crystals require special treatment compared to crystals of small molecules, because macromolecular crystals have a liquid content ranging from approximately 5 to 70%. The current most widely used technique is the loop method, wherein a loop is used to suspend a crystal by surface tension in a thin film of cryoprotected buffer. The first loops were made of gold-plated tungsten wire. These metal loops were replaced by loops made from various fine (10-50 xcexcm diameter) fibers that do not absorb and scatter X-rays to the same extent as metal, such as hair, fibers of glass, nylon, rayon, fly-fishing threads, unwaxed dental floss, cotton, surgical thread and mohair wool.
There are several ways of connecting the loop-supporting pin to the head of the positioning device. Two widely used methods are insertion of a pin directly into the hole in the head of the positioning device and attachment of a magnet to the head of the positioning device, to which a magnetic pin-holder is attracted and rigidly held.
Evaporation from the film suspended in the loop is very rapid because of its large surface-to-volume ratio. Therefore, one of the most critical parameters in a cryocrystallographic experiment is the time between picking up the crystal and flash cooling it. This time should be as short as possible, ideally less than one second, otherwise the crystal can dehydrate or components of the buffer can precipitate. According to Garman et al., all manipulations and motions should be practised on several dry runs with nothing in the loop, to ensure smooth and rapid operation later on. No time should be wasted in viewing the crystal within the loop, since flash cooling an empty loop is less harmful than losing crystals before cooling by stopping to check whether they really are in the loop.
For most protein crystals, flash cooling in a gas stream is perfectly adequate and represents the safest and simplest option. From a practical standpoint, for gas-stream flash cooling it is helpful at first to have a second operator present who can divert the cold gas stream by holding a piece of card over it as soon as the xe2x80x9cfisherxe2x80x9d signals that the crystal is caught. Once the crystal is positioned, the card is then swiftly whipped away ensuring rapid and reproducible cooling. Experienced cryocrystallographers tend to divert the cold gas stream themselves or do not divert it at all while placing the crystal on the head of the positioning device, success depending on the quickness and certainty of their action.
The most common difficulty experienced by experimenters starting to use cryotechniques is ice around, near, on, and/or in the crystal. There are several reasons for ice forming around the crystal. The end of the cryonozzle may be positioned too far from the crystal: ideally it should be as close as possible since the temperature profile of the cold nitrogen stream is very sharp (the temperature rises from 100xc2x0 K. to room temperature over a few millimeters for most open-flow systems). In addition, further away from the nozzle the gas stream becomes dissipated and is thus more susceptible to the effects of turbulence and drafts. If placing the cryonozzle near the crystal results in a shadow on the X-ray detector, thought should be given to changing the angle of approach of the stream. If this proves impossible, the shadow can be masked out during data processing.
A question that often arises concerns the optimum angle of incidence of the cold stream on the crystal. This is not an important factor in a draft-free and carefully monitored experiment. However, most cold streams operate better with the gas flowing downwards. Also, experimental constraints must be taken into account. For instance, for crystal storage enough space must be available to allow cryovial access.
In general, a major reason for ice formation is turbulent flow at the boundaries between the cold gas and warm coaxial stream and between the latter and warm wet air in the room. To prevent this and to allow the desired laminar flow, the flow velocities of the cold and the warm dry gases must be matched. To match the flows, the relative areas of the two gas streams can be calculated and the rates scaled accordingly.
Many classes of biological molecules can be studied by X-ray crystallography, including, but not limited to, proteins, DNA, RNA, and viruses. Scientists have reported the crystal structures of molecules that carry ligands within their receptors, i. e., ligand-receptor complexes.
Given a representation of a target molecule or ligand-receptor complex, scientists can search for pockets or receptors where biological activity can take place. Then scientists can experimentally or computationally design high-affinity ligands (or drugs) for the receptors. Computational methods have alternatively been used to screen for the binding of small molecules. However, these previous attempts have met with limited success. Several problems plague ligand design by computational methods. Computational methods are based on estimates rather than on exact determinations of the binding energies, and rely on simple calculations when compared with the complex interactions that exist within a biomolecule. Moreover, computational models require experimental confirmation, which often expose the models as false positives that do not work on the actual target.
It has recently been discovered that X-ray crystallography can be used to screen compounds that are not known ligands of a target biomolecule for their ability to bind the target. The method comprises the steps of obtaining a crystal of a target biomolecule, exposing the target to one or more test samples that are potential ligands of the target, and determining whether a ligand/biomolecule complex is formed. The target is exposed to potential ligands by various methods, including but not limited to, soaking a crystal in a solution of one or more potential ligands or co-crystallizing a biomolecule in the presence of one or more potential ligands. Structural information from the ligand-receptor complexes found can be used to design new ligands that bind tighter, bind more specifically, have better biological activity or have better safety profiles than known ligands.
According to this novel method, ligands for a target molecule having a crystalline form are identified by exposing a library of small molecules, either singly or in mixtures, to the target (e. g., protein, nucleic acid, etc.). Then, one obtains crystallographic data to compare the electron density map of the putative target-ligand complex with the electron density map of the target biomolecule. The electron density map simultaneously provides direct evidence of ligand binding, identification of the bound ligand, and the detailed three-dimensional structure of the ligand-target complex. Binding may also be monitored by changes in individual reflections within the crystallographic diffraction pattern which are known to be sensitive to ligand binding at the active site. This could serve as a pre-screen but would not be the primary method of choice because it provides less detailed structural information.
By observing changes in the level of ligand electron density or the intensity of certain reflections in the diffraction pattern as a function of ligand concentration either added to the crystal or in co-crystallization, one may also determine the binding affinities of ligands for biomolecules. Binding affinities may also be obtained by competition experiments. Here, the new compound(s) are soaked or co-crystallized with one of a series of diversely-shaped ligands of known binding affinity. If the known ligand appears in the electron density map, the unknown ligands are weaker binders. However, if one of the new compounds is found to compete for the site, it would be the tighter binder. By varying the concentration or identity of the known ligand, a binding constant for the hit may be estimated.
Screening requires exposing a target molecule to thousands of compounds singly or in mixtures. Screening by means of X-ray crystallography requires examining many crystals, which in turn can involve many days of operating 24 hour per day. Such thorough screening can only be accomplished by means of an automated system for mounting crystals onto the X-ray instrument and for aligning the crystals to the X-ray beam.
The use of cryogenic techniques brings great advantages to the crystallographer. One advantage is that the great reduction in radiation damage to crystals at cryogenic temperatures gives the crystallographer effectively infinite crystal lifetimes on an in-house source and vastly extended lifetimes on a synchrotron. Another advantage of cryogenic data collection is that the crystal mounting methods used are mechanically gentler and involve less sample handling. A third advantage of the technique is the facility for in-house screening of flash-cooled crystals and the possibility of storing and transporting them.
The major problem with the use of cryogenic techniques is the high expense of trained operators to mount the samples and collect the data. Therefore, it would be desirable to develop a method for collecting X-ray crystallographic data automatically, without the necessity of a trained operator being present.
Synchrotron X-radiation has become a very common source of X-rays for examining crystals of all types of molecules, small and macromolecular. Because of the particularly intense X-rays available at a synchrotron, cryocooling of samples is usually desirable and often necessary. Although the intense X-rays result in a large reduction in data collection times, often as low as minutes, safety issues complicate sample loading so that the steps of crystal mounting and alignment to the X-ray beam often take as long as or even longer than the data collection step itself. The duration of these mounting and alignment steps results in a significant lowering of efficiency in the use of synchrotron beamlines, which are in great demand and expensive to construct and operate. An automated device for mounting crystal samples onto the X-ray instrument and for accurately aligning the crystal samples to the X-ray beam would significantly reduce the time required for sample loading and greatly accelerate the process of examining a great number of samples. Thus, an automated device would achieve a significant increase in efficiency in the use of synchrotron beamlines.
In one aspect, this invention provides a method for mounting a sample comprising a crystal for X-ray crystallographic analysis, which method comprises the steps of:
(a) providing a crystal holder containing at least a crystal;
(b) providing a tool capable of retrieving the crystal holder, the tool movable by means of a robot;
(c) providing a positioning device for mounting the crystal holder so that the crystal is in the path of a beam of X-rays; and
(d) activating the robot so that the tool retrieves the crystal holder, transfers the retrieved crystal holder to the positioning device, and mounts the transferred crystal holder on the positioning device.
In another aspect, this invention provides a method for aligning a sample comprising a crystal for X-ray crystallographic analysis, which sample is mounted on a positioning device. The method comprises the steps of:
(a) providing a sample, the sample mounted on a positioning device;
(b) providing an apparatus capable of viewing the mounted sample, whereby the apparatus is capable of imaging said mounted sample and determining coordinates of the sample relative to a reference position;
(c) providing a source of power for adjusting the positioning device linearly along three orthogonal axes and rotationally about one of the three axes; and
(d) activating the source of power to cause the positioning device to be adjusted such that the sample is positioned into the path of a beam of X-rays, the adjustments of the positioning device being at a plurality of angles, such that the sample is positioned within the beam of X-rays at any angle of rotational adjustment.
In still another aspect, this invention provides a method for determining the structure of a sample containing a crystal by means of X-ray crystallography, which method comprises the steps of:
(a) providing a crystal holder containing at least a crystal;
(b) providing a tool capable of retrieving the crystal holder, the tool movable by means of a robot;
(c) providing a positioning device for mounting the crystal holder so that the crystal is in the path of a beam of X-rays;
(d) activating the robot so that the tool retrieves the crystal holder, transfers the retrieved crystal holder to the positioning device, and mounts the transferred crystal holder on the positioning device;
(e) providing an apparatus capable of viewing the mounted sample, whereby the apparatus is capable of imaging said mounted sample and determining coordinates of the sample relative to a reference position;
(f) providing a source of power for adjusting the positioning device linearly along three orthogonal axes and rotationally about one of the three axes;
(g) activating the source of power to cause the positioning device to be adjusted such that the sample is positioned into the path of a beam of X-rays, the adjustment of the positioning device being at a plurality of angles, such that the sample is positioned within the beam of X-rays at any angle of rotational adjustment;
(h) providing a beam of X-rays, the beam aimed at the sample; and
(i) recording scattering of X-rays from the sample.
In still another aspect, this invention provides a device for holding a crystal comprising:
(a) a base;
(b) an attachment element projecting from the base;
(c) a stem projecting from the attachment element, the stem supporting a loop for holding the crystal; and
(d) at least one aperture in the attachment element for allowing venting of the device, the device capable of being attached to both a storage cell and a positioning device.
In still another aspect, this invention provides an apparatus for retrieving a crystal holder from a storage cell comprising:
(a) a rotatable element capable of retrieving the crystal holder from the storage cell;
(b) a means for rotating a rotatable element in a given direction of rotation when the rotating means is in a locked mode;
(c) a means for providing a controlled amount of torque when the rotating means is slipping relative to the rotatable element; and
(d) a means for activating the rotating means and the torque controlling means.
In a preferred embodiment, an apparatus for retrieving the crystal holder from the storage cell comprises:
(a) a clutch having a cylindrical housing, the housing comprising a bore surrounded by a wall;
(b) a cylindrical plunger capable of moving axially within the bore of the housing;
(c) the plunger having at least one elongated grove on the exterior periphery thereof, the groove capable of receiving a locking pin;
(d) the housing having at least one aperture extending through the wall thereof;
(e) at least one spring pin retained in the aperture, the pin capable of engaging the elongated groove when the plunger is disposed in a first position in the housing, the pin capable of disengaging the elongated groove when the plunger is disposed in a second position in the housing;
(f) a means in the housing for resiliently biasing the plunger toward the first position in the housing;
(g) a friction plate in contact with the interior wall of the housing, the friction plate providing friction between an output flange and the friction plate; and
(h) a shaft attached to the plunger, the shaft capable of transmitting torque to the friction plate, the shaft further capable of moving axially with respect to the friction plate.
In still another aspect, this invention provides a device for holding a plurality of samples, the device comprising a plurality of storage cells. The device is capable of maintaining the temperature of the samples at a temperature of not greater than about 160xc2x0 K. Each of the storage cells has a guided passageway; the guided passageway has a base at the lower end thereof and an opening at the upper end thereof. The area of the opening is greater than the area of the base. At least one side-wall circumscribes the base and the opening. The base is of sufficient area to allow placement of a sample holder. The opening is of sufficient area to allow ingress of a tool for retrieving the sample holder. The base has attached thereto a means for locking the sample holder to the sample-holding device. The device may also be equipped with a lid that can be moved by means of a robot.
This invention also provides various tools and auxiliary devices for carrying out the methods described herein.
This invention provides numerous advantages over conventional methods of X-ray crystallography. First, this invention makes it possible to reduce the number of trained operators required to conduct X-ray analysis of crystals. Second, this invention makes it possible to analyze crystals without the need for an operator to be present. Third, this invention makes it possible to increase the speed of analysis by X-ray crystallography, thereby increasing the throughout of the analysis. Fourth, this invention makes it possible to standardize the handling of samples and reduce the possibility of errors by the operator. This invention also facilitates collection of data 24 hours per day, seven days per week, thereby increasing the utilization of expensive X-ray crystallography equipment. This invention further facilitates the retrieval and preservation of crystal samples after data has been collected, thereby making it possible to reanalyze the sample at a later date.